EP1109926B1 - Kondensierte plasmid-liposome komplex zur transfektion - Google Patents

Kondensierte plasmid-liposome komplex zur transfektion Download PDF

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EP1109926B1
EP1109926B1 EP98949342A EP98949342A EP1109926B1 EP 1109926 B1 EP1109926 B1 EP 1109926B1 EP 98949342 A EP98949342 A EP 98949342A EP 98949342 A EP98949342 A EP 98949342A EP 1109926 B1 EP1109926 B1 EP 1109926B1
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plasmid
liposome
lipid
complexes
composition according
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EP1109926A1 (de
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Shi Kun Huang
Edwin Kiyoshi Oto
Mohammad Hassanipour
Bei Jin
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Alza Corp
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Alza Corp
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to an improvement in a method for preparing plasmid-liposome complexes for in vivo transfection of a gene and to compositions prepared by the method.
  • a variety of methods have been developed to facilitate the transfer of genetic material into specific cells, e.g., gene therapy. These methods are useful for both in vivo or ex vivo gene transfer.
  • a gene is directly introduced (intravenously, intraperitoneally, aerosol, etc.) into a subject.
  • ex vivo (or in vitro) gene transfer the gene is introduced into cells after removal of the cells from specific tissue of an individual. The transfected cells are then introduced back into the subject.
  • Delivery systems for achieving in vivo and ex vivo gene therapy include viral vectors, such as a retroviral vector or adenovirus vectors, microinjection, electroporation, protoplast fusion, calcium phosphate, and liposomes (Felgner, et al., 1987; Mulligan, 1993; Morishita, et al., 1993).
  • Liposomal mediated gene therapy has, for example, involved the use of cationic liposomes formed from LIPOFECTIN , a reagent consisting of a cationic lipid and a neutral lipid (Felgner, et al., 1989, 1993).
  • Other liposomal-mediated methods of gene therapy have been described (Trubetskoy, et al., 1992; Morishita, et al., 1993; Rose, 1994), where electrostatic complexes of cationic liposomes and DNA are formed.
  • Liposome-based transfection compositions have included a polycation, such as protamine or polylysine, to bind the DNA to the lipid particles (Wagner, et al ., 1991) or to condense the DNA (Gao and Huang, 1996; Li and Huang, 1996).
  • a polycation such as protamine or polylysine
  • liposomal-mediated gene therapy methods and compositions described to date have recognized limitations, including, for example, the toxicity of LIPOFECTIN, the large size of the DNA-liposome complexes, and rather poor in vivo transfection efficiencies.
  • composition of plasmid-liposome complexes for use in transfecting a host cell with a gene contained in a plasmid comprising condensed plasmid molecules, said molecules condensed with a polycationic condensing agent and suspended in an aqueous medium containing a non-electrolyte solute, said medium having an ionic strength less than the ionic strength of a 10mM solution containing a monovalent ionizable salt, and cationic liposomes comprising a diacyl chain cationic vesicle-forming lipid, dimethyldioctadecylammonium bromide (PDAB), N-[1-(2,3,-ditetradecyloxy)propyl ⁇ -N,N-dimethyl-N-hydroxyethylammonium bromide (OMRIE), N-[1-(2,3,-dioleyloxy)propyl]-N,N-d
  • PDAB dimethyldio
  • the condensed plasmid molecules are DNA plasmid molecules containing a gene selected from the group consisting or genes encoding for cystic fibrosis transmembrane conductance regulator, Factor VIII, interleukin-2 or p53.
  • the condensing agent is a polycation selected from histones, poly-1-glutamine, protamine, melittin and polymyxin B.
  • the condensing agent is a histone selected from total histone, histone 1 and histone 4.
  • the ratio of liposome lipid to plasmid is between 8-10 nmole liposome lipid/ ⁇ g plasmid.
  • non-electrolyte osmotic solute is glucose, sucrose or dextran.
  • the cationic liposomes may comprise a cationic vesicle-forming lipid selected from dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleyloxy-3-(trimethylamino)propane (DOTAP), N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-(2,3,-dioleyloxylpropyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), N-[1-(2,3-dioleyloxy)propyl]-N,N,N,-trimethylammonium chloride (DOTMA).
  • DDAB dimethyldioctadecylammonium bromide
  • DOTAP 1,2-dioleyloxy-3-(trimethylamino)propane
  • the cationic liposomes further include a neutral vesicle-forming lipid
  • the cationic liposomes further include cholesterol.
  • the cationic liposomes have a surface coating of hydrophilic polymer chains by including a vesicle-forming lipid derivatized with such a hydrophilic polymer,
  • at least a portion of the hydrophilic polymer chains are joined to the vesicle-forming lipid by a releasable bond, e . g ., a bond effective to release the hydrophilic polymer chains in response to an existing or an induced physiologic condition.
  • the plasmid-liposome complexes can further include a ligand attached to distal ends of the hydrophilic polymer chains for ligand-specific binding to a receptor molecule on a target cell surface.
  • the hydrophilic polymer is polyethyleneglycol.
  • the present invention also provides a method of preparing plasmid-liposome complexes comprising the steps of;
  • the plasmid-liposome complexes of the invention may be used for transfecting a host cell with a gene contained in a DNA plasmid, where the DNA plasmid contains a gene selected from the group consisting of genes encoding for Factor VIII, interleukin-2 or p53.
  • the plasmid-liposome complex is for use in transfecting a host cell in the lung of a subject with a DNA plasmid containing cystic fibrosis transmembrane conductance regulator or, for lung carcinomas, cytokines, such as interleukin-2, or tumor suppressor genes, such as p53.
  • the present invention is directed to a plasmid-liposome complex, and to an improvement in a method of preparing such a complex, for use in in vivo transfection of a host cell.
  • the composition includes plasmid molecules which are condensed with a polycationic condensing agent and then suspended in low-ionic strength medium.
  • the condensed plasmid molecules are mixed with lipid particles, such as liposomes, to form plasmid-liposome complexes.
  • the improvement in the method of preparation as will be described, relates to selection of the condensing agent, selection of the suspension medium and selection of the liposome lipid to plasmid ratio.
  • the plasmid-liposome complex includes condensed plasmid molecules and liposomes.
  • Liposomes refer to lipid vesicles having an outer lipid shell, typically formed on one or more lipid bilayers, encapsulating an aqueous interior.
  • the liposomes are cationic liposomes composed of between about 20-80 mole percent of a cationic vesicle-forming lipid, with the remainder neutral vesicle-forming lipids and/or other components.
  • vesicle-forming lipid refers to any amphipathic lipid having hydrophobic and polar head group moieties and which by itself can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids.
  • a preferred vesicle-forming lipid is a diacyl-chain lipid, such as a phospholipid, whose acyl chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • a cationic vesicle-forming lipid is one whose polar head group with a net positive charge, at the operational pH, e.g., pH 4-9.
  • Typical examples include phospholipids, such as phosphatidylethanolamine, whose polar head groups are derivatized with a positive moiety, e.g., lysine, as illustrated, for example, for the lipid DOPE derivatized with L-lysine (LYS-DOPE) (Guo, et al., 1993).
  • the glycolipids such as cerebrosides and gangliosides having a cationic polar head-group.
  • cationic vesicle-forming lipid which may be employed is cholesterol amine and related cationic sterols.
  • exemplary cationic lipids include 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE); N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOMA); and dimethyldioctadecylammonium bromide (DDAB).
  • DOTAP 1,2-dioleyloxy-3-(trimethylamino) propane
  • DMRIE N-[1-(2,3,-
  • the remainder of the liposomes are formed of neutral vesicle-forming lipids; meaning vesicle forming lipids which have no net charge or which may include a small percentage of lipids having a negative charge in the polar head group.
  • lipids include the phospholipids, such as phosphatidylcholine (PC), phosphatidyl ethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), and cholesterol, cholesterol derivatives, and other uncharged sterols.
  • lipids can be obtained commercially, or prepared according to published methods.
  • Other lipids that can be included in the invention are glycolipids, such as cerebrosides and gangliosides.
  • the plasmid-liposome complex includes liposomes having a surface coating of hydrophilic polymer chains, effective to extend the blood circulation time of the plasmid/liposome complexes.
  • Suitable hydrophilic polymers include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinyl-pyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethyl-cellulose.
  • a preferred hydrophilic polymer chain is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 500-10,000 daltons, more preferably between 1,000-5,000 daltons.
  • the hydrophilic polymer may have solubility in water and in a non-aqueous solvent, such as chloroform.
  • the coating is preferably prepared by including in the vesicle-forming lipids forming the liposomes, between 1-20 mole percent of a vesicle-forming lipid, preferably a phospholipid or other diacyl-chain lipid, derivatized at its head group with the polymer chain. Exemplary methods of preparing such lipids, and forming polymer coated liposomes therewith, have been described in U.S. Patents Nos. 5,013,556, and 5,395,619.
  • hydrophilic polymer can be stably coupled to the lipid, or coupled through an unstable linkage which allows the polymer-coated plasmid-liposome complexes to shed or "release" the hydrophilic polymer coating during circulation in the bloodstream or after localization at a target site.
  • Attachment of hydrophilic polymers, in particular polyethyleneglycol (PEG), to vesicle-forming lipids through a bond effective to release the polymer chains in response to a stimulus have been described, for example in WO 98/16202 and WO 98/16201, and by Kirpotin, D. et al. ( FEBS Letters, 388 :115-118 (1996).
  • the releasable linkage in one embodiment, is a chemically releasable linkage which is cleaved by administration of a suitable releasing agent or is cleaved under selective physiological conditions, such as in the presence of enzymes or reducing agents.
  • a suitable releasing agent for example, ester and peptide linkages are cleaved by esterase or peptidase enzymes.
  • Disulfide linkages are cleaved by administration of a reducing agent, such as glutathione or ascorbate, or by a reducing agent present in vivo, such as cysteine, which is present in plasma and intracellularly.
  • releasable linkages include pH sensitive bonds and bonds which are cleaved upon exposure to glucose, light or heat.
  • the hydrophilic polymer chains can be attached to the liposome by a pH sensitive bond, and the plasmid-liposome complexes are targeted to a site having a pH effective to cleave the bond and release the hydrophilic chains, such as a tumor region.
  • Exemplary pH sensitive bonds include acyloxyalkyl ether, acetal and ketal bonds.
  • the cleavable bond is a disulfide bond, broadly intended herein to refer to sulfur-containing bonds.
  • Sulfur-containing bonds can be synthesized to achieve a selected degree of lability and include disulfide bonds, mixed sulfide-sulfone bonds and sulfide-sulfoxide bonds. Of the three bonds, the disulfide bond is least susceptible to thiolysis and the sulfide-sulfoxide bond is most susceptible.
  • Such releasable bonds are useful to tailor the rate of release of the hydrophilic polymer segment from the plasmid-liposome complexes.
  • a very labile disulfide bond can be used for targeting to blood cells or endothelial cells, since these cells are readily accessible and a shorter liposome blood circulation lifetime is sufficient.
  • a long-lasting or hearty disulfide bond can be used when the target is tumor tissue or other organs where a longer liposome blood circulation lifetime is generally needed for the complexes to reach the desired target.
  • the releasable bond attaching the hydrophilic polymer chains to the liposome is cleaved in vivo typically as a result of change in environment, such as when the liposomes reach a specific site with a slightly lower pH, such as a region of tumor tissue, or a site with reducing conditions, such as a hypoxic tumor. Reducing conditions in vivo can also be effected by administration of a reducing agent, such as ascorbate, cysteine or glutathione.
  • the cleavable bond may also be broken in response to an external stimuli, such as light or heat.
  • the plasmid-liposome complexes include an affinity moiety or targeting ligand effective to bind specifically to target cells at which the therapy is aimed.
  • Such moieties can be attached to the surface of the liposome or to the distal ends of hydrophilic polymer chains.
  • Exemplary moieties include antibodies, ligands for specific binding to target cell surface receptors and the like, as described, for example, in PCT application Nos. WO US94/03103, WO 98/16202 and WO 98/16201.
  • the moiety can also be a hydrophobic segment to facilitate fusion of the complex with a target cell.
  • This section describes the preparation of the condensed-phase plasmid employed in the plasmid-liposome complex of the invention.
  • Polycationic condensing agents used to condense the plasmid are multiply charged cationic polymers, typically biopolymers such as such as spermidine, spermine, polylysine, protamine, total histone, specific histone fractions such as H1, H2, H3, H4, and other polycationic polypeptides, but may also include biocompatible polymers, such as polymyxin B. It will be appreciated that these polycationic condensing agents can be used in free base or salt forms, for example, protamine sulfate and polylysine hydrobromide.
  • the polycationic condensing agent is a histone, which, as referred to herein, includes total histone or specific histone fractions.
  • Plasmids suitable for use in the complex are preferably circularized or closed double-stranded molecules having sizes preferably in the 5-40 Kbp (kilo basepair) range.
  • the plasmids are constructed according to well-known methods and include a therapeutic gene, i.e., the gene to be expressed in gene therapy, under the control of suitable promoter and terminator control elements, and other elements necessary for replication within the host cell and/or integration into the host-cell genome. Methods for preparing plasmids useful for gene therapy in genes or other mammals are widely known and referenced.
  • the genes to be introduced for gene therapy by the complex of the invention generally fall into one of three categories:
  • genes which are intended to overcome a gene deficiency or defect in the subject i.e., where the subject fails to produce active, endogenous protein at all or within normal levels, and the gene introduced in the plasmid is intended to make up this deficiency.
  • genes encoding adenosine deaminase include genes encoding adenosine deaminase (ADA), for gene expression in stem cells or lymphocytes; genes encoding purine nucleoside phosphorylase deficiency, deficiency in prostaglandin G/H synthase, therapy of Lesch-Nyhan syndrome caused by a deficiency in hypoxanthine-guanine phosphoribosyltransferase, genes encoding a variety of circulating proteins, such as ⁇ 1 -antitrypsin, clotting factors (e.g., Factor VIII, Factor IX) and globins (e.g., ⁇ -globin, hemoglobin), for the treatment of hemophilia, sickle-cell anemia and other blood-related diseases, and genes encoding hormones and other peptide regulators.
  • ADA adenosine deaminase
  • polypeptides designed to treat any existing pathology, such as cancer, or a pathogenic condition such as viral infection examples include gene therapy to supply the p53 gene for cancer therapy, the gene for the CD4 peptide to inhibit HIV infection, the gene for the Pseudomonas peptide to inhibit binding of Pseudomonas to epithelial cells, and specific antibody genes to inhibit a targeted pathogen.
  • the third class includes genes intended to produce an mRNA transcript that can act as an antisense molecule to inhibit an undesirable protein expression, such as overexpression of proteins specific for tumor growth, or expression of viral proteins.
  • a plasmid-liposome complex formed by mixing condensed-phase plasmid and cationic liposomes, in an aqueous medium containing a non-electrolyte solute, said medium having an ionic strength less than the ionic strength of a 10mM solution containing a monovalent ionizable salt, produces complexes having a substantially homogeneous size of typically less than 200 nm and preferably in the range of 50-200 nm, more preferably between 100-200 and most preferably between 120-180 nm.
  • the complexes are further characterized by a retention of activity of the expression of the encoded gene, that is the complexes have a transfection stability. This is evidenced by the ability of the complexes after storage for 30 days, and preferably 90 days, at 4 °C, to transfect cells and achieve expression of the encoded gene, as will be discussed below.
  • the condensed-phase plasmid is formed by adding to the plasmid, in an aqueous medium containing a non-electrolyte solute, said medium having an ionic strength less than the ionic strength of a 10 ⁇ M solution containing a monovalent ionizable salt, a polycationic polymer condensing agent of the type identified above.
  • the cationic polymer is added to the plasmid solution to a preferred concentration at which charge stoichiometry is achieved, i.e.
  • the weight ratio of added DNA plasmid to added polymer is between about 0.1-5.0, more preferably, between 0.3-2.0.
  • the condensing agent is preferably added slowly to the plasmid suspension with stirring, e.g., over a 10 minute period.
  • the ratio of liposome lipids to plasmid is an important parameter for achieving maximum transfection. That ratio, in nmole liposome lipid/ ⁇ g plasmid is greater than 5 but less than 25, preferably greater than 8 but less than 18, more preferably greater than 10 but less than 15 and most preferably between 12-14.
  • a critical feature of the invention is the mixing of liposomes and condensed phase plasmid in said aqueous medium.
  • the final concentration of the medium, including ions present in the DNA, condensing agent, and liposome lipid species, is less than the ionic strength of a 10 mM monovalent ionizable salt, such as NaCl, and preferably less than about 1 mM.
  • a 10 mM monovalent ionizable salt such as NaCl
  • low ionic strength is readily obtained by employing free base or free acid plasmid, polycation, and lipid species, removing electrolyte components, or alternatively, employing sufficiently dilute concentrations of the components to maintain a low ionic strength, and/or removing electrolytes generated from the components by dialysis or the like.
  • the composition is prepared in the presence of a non-electrolyte solute such as glucose to provide an osmotic balance as an injectable formulation.
  • Fig. 4 shows that the complexes as prepared in Example 1 form a homogeneous population having an average size of about 146 nm (standard deviation of 45 nm).
  • This and other studies performed in support of the invention indicate that the composition when prepared according to the described method achieves complexes have a homogeneous population, as evidenced by a single peak indicating a population of complexes having a relatively narrow size distribution.
  • the complexes have a size of less than 200 nm, more preferably between 50-200 nm, most preferably between 100-200 and still more preferably between 120-180 nm.
  • the plasmid-liposome complexes are stable, that is, the complexes maintain their initial size, e.g., there is little aggregation of the complexes, and the complexes retain therapeutic activity, for at least 30 days after storage at 4 °C.
  • Fig. 5 provides evidence of complex size stability and shows the complex size, as measured by dynamic light scattering, as a function of time.
  • a suspension of the plasmid-liposome complexes in water/glucose were stored at 4 °C and analyzed after 7, 14, 21, 28, 60 and 90 days of storage. Initially after complex formation, the average complex size was about 150 nm and, as seen, after 90 days of storage, the complex size remained at about 150 nm.
  • Complex stability with respect to retention of therapeutic activity is discussed below in Figs. 11A-11B.
  • Plasmid-liposome complexes prepared in accordance with the invention were administered to mice to determine the transfection efficiency of various plasmid-liposome complex formulations and to determine stability of transfection, biodistribution of the complex, pharmacokinetics and dose response.
  • the exemplary in vivo transfection procedure is described in Example 2.
  • Fig. 6 shows the percent of injected dose as a function of time after intravenous injection. Immediately after injection of the plasmid-liposome complex, about 7% of the injected dose is in the blood stream. Other studies have determined that the remaining percentage of the complexes localize in the lung immediately after injection. After a period of time the complexes are neutralized by serum proteins in the lung and enter the blood stream, as evidenced by the increase in the percentage of injected dose to about 22% at the 5 hour time point (Fig. 6). The complexes are then cleared from the bloodstream, with about 12% of the injected dose present at 24 hours.
  • Plasmid-liposome complexes were prepared for in vivo administration with varying ratios of liposome lipid to plasmid.
  • the complexes were prepared according to the general procedure set forth in Example 1 by varying the amount of polycation condensing agent, and the total amount of liposome lipids.
  • the amount of polycation condensing agent varied between 100-500 ⁇ g and the liposome lipid/plasmid ratio varied between 8-18 nmoles lipid/ ⁇ g plasmid.
  • Figs. 7-9 show the results for plasmid-liposome complexes prepared with total histone (Figs. 7A-7E), histone H1 (Figs. 8A-8E) and histone H4 (Figs. 9A-9E) as the polycationic condensing agents.
  • Figs. 7A-7E total histone
  • Figs. 8A-8E histone H1
  • Figs. 9A-9E histone H4
  • Table 1 summarizes the compositions for the plasmid-liposome complexes prepared using total histone as the polycationic condensing agent.
  • the amount of total histone was varied between 100-500 ⁇ g to vary the ratio of plasmid/total histone from 0.2-1.0.
  • the ratio of liposome lipids/plasmid was also varied and ratios of 8, 14 and 18 nmoles liposome lipids/ ⁇ g plasmid were tested.
  • Table 1 Component Formulation Number 1 1 2 3 4 5 6 pNSL plasmid, ⁇ g 100 100 100 100 100 100 total histone, ⁇ g 200 100 200 350 500 200 ⁇ moles liposome lipids 2 0.8 1.4 1.4 1.4 1.4 1.8 ⁇ g plasmid/ ⁇ g total histone 0.5 1.0 0.5 0.3 0.2 0.5 moles lipids/ ⁇ g plasmid 8 14 14 14 14 18 1 In vivo results for each formulation number shown in Figs. 7A-7E. 2 Liposome prepared from 1:1 mole ratio of DDAB:cholesterol.
  • Figs. 7A-7E The results of in vivo administration in mice of the formulations summarized in Table 1 are shown in Figs. 7A-7E, where luciferase expression, in relative light units (RLU)/10 seconds/mg protein, is shown in the lung (Fig. 7A), liver (Fig. 7B), heart (Fig. 7C), spleen (Fig. 7D) and kidney (Fig. 7E).
  • the figures indicate that there is a window where the transfection is highest. Specifically, for total histone as the condensing agent, the transfection is highest where the liposome lipid/plasmid ratio is greater than 8 nmoles lipid/ ⁇ g plasmid and less than 18 nmoles lipid/ ⁇ g.
  • Table 2 summarizes the plasmid-liposome complex compositions prepared and tested in vivo using histone H1 as the polycationic condensing agent.
  • the ratio of plasmid/histone H1 ratio was 0.3 or 0.5 and the liposome lipid/plasmid ratio was varied from 8, 14 and 18 nmoles lipid/ ⁇ g plasmid.
  • Table 2 Component Formulation Number 1 7 8 9 10 11 pNSL plasmid, ⁇ g 100 100 100 100 100 histone H I, ⁇ g 350 200 350 200 350 ⁇ moles liposome lipids 2 0.8 1.4 1.4 1.4 1.4 ⁇ g plasmid/ ⁇ g histone H1 0.3 0.5 0.3 0.5 0.3 nmoles lipids/ ⁇ g plasmid 8 14 14 18 18 1 ln vivo results for each formulation number shown in Figs. 8A-8E. 2 Liposomes prepared from 1:1 mole ratio of DDAB:cholesterol.
  • Figs. 8A-8E The results of in vivo administration in mice of the formulations summarized in Table 2 are shown in Figs. 8A-8E, where luciferase expression, in relative light units (RLU)/10 seconds/mg protein, is shown in the lung (Fig. 8A), liver (Fig. 8B), heart (Fig. 8C), spleen (Fig. 8D) and kidney (Fig. 8E).
  • RLU relative light units
  • Tables 3A and 3B summarize the formulations of plasmid-liposome complexes formed using histone H4 as the polycationic condensing agent.
  • the plasmid/histone H4 ratio varied from 0.2, 0.3 or 0.5 and the liposome lipid/plasmid ratio was varied from 8, 14 or 18 nmoles lipid/ ⁇ g plasmid.
  • Table 3A Component Formulation Number 1 12 13 14 15 pNSL plasmid, ⁇ g 100 100 100 100 histone H4, ⁇ g 200 350 500 200 ⁇ moles liposome lipids 2 0.8 0.8 0.8 1.4 ⁇ g plasmid/ ⁇ g histone H4 0.2 0.3 0.2 0.5 nmoles lipids/ ⁇ g plasmid 8 8 14 1 In vivo results for each formulation number shown in Figs. 9A-9E. 2 Liposomes prepared from 1:1 mole ratio of DDAB:cholesterol.
  • Table 3B Component Formulation Number 1 16 17 18 19 20 pNSL plasmid, ⁇ g 100 100 100 100 100 histone H4, ⁇ g 350 500 200 350 500 ⁇ moles liposome lipids 2 1.4 1.4 1.8 1.8 1.8 ⁇ g plasmid/ ⁇ g histone H4 0.3 0.2 0.5 0.3 0.2 nmoles lipids/ ⁇ g plasmid 14 14 18 18 18 1 In vivo results for each formulation number shown in Figs. 9A-9E. 2 Liposomes prepared from 1:1 mole ratio of DDAB:cholesterol.
  • Figs. 9A-9E The results of in vivo administration in mice of the formulations summarized in Tables 3A and 3B are shown in Figs. 9A-9E, where luciferase expression, in relative light units (RLU)/10 seconds/mg protein, is shown in the lung (Fig. 9A), liver (Fig. 9B), heart (Fig. 9C), spleen (Fig. 9D) and kidney (Fig. 9E).
  • RLU relative light units
  • plasmid-liposome complexes were prepared using poly-1-glutamine, melittin (a low molecular weight peptide containing 26 amino acids) or polymyxin B sulfate as polycationic condensing agents. Each of these are commercially available from Sigma Chemical Co.
  • the complexes were injected in mice, as described Example 2, and in vivo transfection was measured by determining luciferase expression.
  • Table 4 summarizes the plasmid-liposome complex compositions prepared using poly-1-glutamine, melittin or polymyxin B as polycationic condensing agents. The amount of condensing agent was varied from 50-200 ⁇ g. Table 4 condensing agent Condensing Agent 1 poly-l-glutamine melittin polymyxin B Formulation No.
  • Figs. 10A- 10E The results of in vivo administration in mice of the plasmid-liposome complexes in Table 4 are shown in Figs. 10A- 10E, where luciferase expression, in pg luciferase/mg protein, is shown in the lung (Fig. 10A), liver (Fig. 10B), heart (Fig. 10C), spleen (Fig. 10D) and kidney (Fig. 10E).
  • the lipid/plasmid ratio for each of the formulations was constant at 14 nmoles lipid/ ⁇ g plasmid, falling about midway in the preferred range of 5-25 nmoles liposome lipid/ ⁇ g plasmid.
  • liposome lipid/plasmid ratio in nmoles lipid/ ⁇ g plasmid
  • the ratio is between 8-18, even more preferably between 10-15 and most preferably between 12-14 nmoles liposome lipid/ ⁇ g plasmid.
  • the plasmid-liposome complex of the present invention is stable, as evidenced by little change in particle size (see Fig. 5), for as long as 90 days at 4° C.
  • the expression stability of the complex was determined by administering plasmid-liposome complexes which were stored at 4 °C to mice. Specifically, the suspension of plasmid-liposome complexes was administered intravenously to mice immediately after preparation of the complex (day 0) and at 7, 14, 21, 28, 30 and 90 days after storage at 4 °C. Following the procedure detailed in Example 2, luciferase expression in the lung and liver was determined, and the results are shown in Figs. 11A-11B.
  • luciferase expression in the lung (Fig. 11A) and the liver (Fig. 11B) remain constant as a function of storage time of the complexes. It is clear that the complex retained the ability to express the encoded gene for the 90 day test period. Accordingly, in one embodiment of the invention that the complex is characterized by the ability to retain more than 50% of the expression activity measured at day 0 for at least 30 days, more preferably for 90 days.
  • a dose-response study was performed using plasmid-liposome complexes prepared as described in Example 1.
  • the plasmid-liposome complex was administered intravenously in mice at five dosage levels of plasmid: 25, 50, 200 250 and 200 ⁇ g.
  • Twenty-four hours after administration luciferase expression in the lung, heart, liver, kidney and spleen was measured, and the results are shown in Fig. 12.
  • the luciferase expression measured was proportional to the dose administered, with the highest expression in the lung.
  • the systemic luciferase expression 24 hours following administration of the plasmid-liposome complex in mice is shown in Fig. 13.
  • the plasmid-liposome complex distributes widely, as evidenced by luciferase expression in the bone marrow, lymph nodes and brain.
  • mice bearing metastatic Lewis lung tumors were treated with plasmid-liposome complexes. After tumor inoculation, the mice were treated with one of eight treatment regimens set forth in Table 5 in Example 3.
  • the treatments included administration of plasmid-liposomes complexes prepared using plasmids carrying genes encoding for p53 (pCMVp53) and interleukin 2 (pCMVIL2) and a combination treatment of ganciclovir and plasmid-liposome complexes prepared with a pHSVtk (Herpes Simplex Virus thymidine kinase) plasmid.
  • a control group of animals received saline, and comparative groups of animals received complexes prepared with the pNSL plasmid encoding for luciferease (Example 1) or ganciclovir alone.
  • Figs. 14A-14C show the percent of surviving animals as a function of days post tumor-cell inoculation.
  • the animals treated with saline are represented with the closed squares. As seen, all of the saline-treated animals died by 24 days after tumor inoculation.
  • the animals treated with complexes including the 50 ⁇ g (solid inverted triangles) and 75 ⁇ g (open squares) pHSVtk plasmid and with ganciclovir are shown. All of the animals treated with ganciclovir alone (open circles) died by day 37. In contrast, the animals treated with the combination therapy of liposome-plasmid complexes and ganciclovir fared better, with 70% of those treated with the higher plasmid dose surviving the study.
  • Fig. 14B shows the percent survival of animals treated with plasmid-liposome complexes including pCMVp53 (solid inverted triangles) and pNSL (encoding for luciferase) (open circles). As seen, 20% of the animals treated with complexes including the plasmid encoding for the p53 gene survived the duration of the study, whereas the saline-treated animals (closed squares) and the animals treated with the pNSL plasmid encoding for luciferase-reporter gene died.
  • Fig. 14C shows the results for animals treated with complexes including the pCMVIL2 plasmid, administered at 50 ⁇ g plasmid DNA (inverted solid triangles) and 75 ⁇ g plasmid DNA (open squares). As seen, between about 15-20% of the animals survived at the end of the treatment period, whereas left untreated (saline control, solid squares) all of the animals died by day 24. Similarly, animals treated with complexes including the luciferase-encoding pNSL plasmid (open circles) died by day 31.
  • mice treated with saline plus symbols
  • mice treated with plasmid-liposome complexes at dosages of 50 ⁇ g plasmid (open circles), 75 ⁇ g plasmid (closed triangles) and 100 ⁇ g plasmid (open, inverted triangles).
  • the pNSL-luciferase-plasmid-liposome complex was administered to mice and luciferase expression in various tissues was examined 24 hours after administration of plasmid-liposome complexes. As seen in Fig. 16, the highest luciferase expression was observed in the lung and in the tumors.
  • Plasmid-liposome complexes prepared in accordance with the method of the invention form a substantially homogeneous population having sizes of less than about 200 nm, as evidenced by dynamic light scattering.
  • the complexes are stable for 90 days, with no aggregation of complexes, as evidenced by dynamic light scattering.
  • the transfection activity of the complexes is also stable, where the complexes retain more than 50% of transfection efficiency after storage for at least 30 days at 4 °C.
  • DDAB Dimethyldioctadecylammonium
  • histone H1 and histone H4 were obtained from Boehringer Mannheim (Indianapolis, IN).
  • Poly-1-glutamine, melittin and polymyxin B sulfate were obtained from Sigma Chemical Co. (St. Louis, MO).
  • Size distribution measurements were obtained by dynamic light scattering (DLS) using a Coulter N4MD instrument, operated according to the manufacturer's instructions. The results were expressed as the mean diameter in nm and standard deviation of a Gaussian distribution of particles by relative volume.
  • a pNSL plasmid encoding for luciferase was constructed from two commercially available plasmids, pGFP-N1 plasmid (Clontech, Palo Alto, CA) and pGL3-C (Promega Corporation, Madison, WI).
  • the pGL3-C was cut with Xba I and blunt-end ligated using the Klenow fragment of E. coli DNA polymerase. It was then cut with Hind III and the 1689-bp fragment, carrying the luciferase gene, was gel-purified.
  • the pGFP-N 1 plasmid was cut with Sma I and Hind III and the 4.7 kb fragment, isolated from an agarose gel, was ligated with the luciferase fragment. JM109 E. coli cells were transformed and 20 colonies were selected; about half of then showed the presence of inserts; 8 clones with inserts were cut with Nam HI and Xho I to further confirm the presence of the luciferase gene; 7 of them were positive.
  • Cationic liposomes were prepared according to standard procedures by dissolving 10 ⁇ mol DDAB and 10 ⁇ mol cholesterol in an organic solvent containing primarily CHCl 3 .
  • the lipids were dried to a thin film by rotation under reduced pressure.
  • the lipid film was hydrated by addition of distilled water to form a suspension of liposomes at a concentration of 20 ⁇ mole/ml.
  • the liposomes were sized by sonication or by sequential extrusion through Nucleopore polycarbonate membranes with pore sizes of 0.4 ⁇ m, 0.2 ⁇ m, 0.1 ⁇ m and 0.05 ⁇ m to obtain liposomes of less than about 200 nm in size (Nucleopore, Pleasanton, CA).
  • the DNA plasmid pNSL encoding for luciferase was condensed according to the following procedure. 400 ⁇ l of the plasmid (1 mg/ml in distilled water) was diluted with 310 ⁇ l distilled water and then mixed with 90 ⁇ l of 50% glucose. 100 ⁇ l of a polycationic condensing agent (total histone, histone H1, histone H4, poly-1-glutamine, melittin or polymyxin B) from a stock solution of 1 mg/ml in distilled water was added to the plasmid solution slowly with stirring. The mixture was stirred for 10 minutes.
  • a polycationic condensing agent total histone, histone H1, histone H4, poly-1-glutamine, melittin or polymyxin B
  • Plasmid-liposome complexes having a liposome lipid/plasmid ratio of 14 nmole lipid/ ⁇ g plasmid was prepared by diluting 280 ⁇ l of the liposome suspending with 350 ⁇ l of distilled water and then adding 70 ⁇ l of 50% glucose. The suspension of condensed plasmids was slowly added to the diluted cationic liposome suspension with continuous stirring for 10 minutes.
  • mice obtained from Simonsen (Gilroy, CA). Each plasmid-liposome complex formulation was injected via tail vein into 3 mice. The mice were sacrificed 24 hours after injection and tissues (lung, liver, spleen, kidney, heart) were collected following perfusion with heparinized PBS (4 C) under anesthesia.
  • Plasmid-liposome complexes were prepared according to the procedure of Example 1 using the following plasmids: pNSL encoding for luciferase, prepared as described in Example 1, and pCMVp53, pCMVIL2 and pHSVtk (all commercially available).
  • mice Eighty B6C3-F1 male mice were obtained from Taconic Farms (German Town, NY) and allowed to acclimate for 3 days prior to initiation of the experiment. Animals were housed in appropriate isolated caging with ad lib sterile rodent food and acidified water and a 12:12 light:dark cycle. Animals were randomized into treatment groups prior to inoculation of tumors based on body weight. Animals were randomized into treatment groups prior to inoculation of tumors.
  • Tumors were inoculated by taking growing Lewis lung tumors from other B6C3-F1 mice by sterile surgical harvest after euthanasia.
  • the tumors were mechanically minced as finely as possible and briefly digested in an enzyme mix of collagenase, protease and DNAse at 37 °C. After digestion and washing in media (RPMI + 15% FCS) cells were counted with a hemocytometer. Cells were spun down and resuspended in media at 10 6 cells per ml (10 5 cells per 0.1 ml injection). The resuspended cells were drawn into individual syringes (0.1 ml, with continuous mixing) for intravenous injection into the tail vein of each animal.
  • Composition 30 1,2,3,4 saline, 0.1 ml IV 31 1 ganciclovir, 100 ⁇ g/kg IP twice daily for 6 days for 5 weeks 32 1 liposome/pHSVtk complex, 50 ⁇ g DNA/mouse + ganciclovir 100 ⁇ g/kg twice daily for 6 days after each administration of the plasmid/liposome complex 33 1 liposome/pHSVtk complex, 75 ⁇ g DNA/mouse + ganciclovir 100 ⁇ g/kg twice daily for 6 days after each administration of the plasmid/liposome complex.
  • Figs. 14A-14C The percent of surviving animals for each treatment group are shown in Figs. 14A-14C. Toxicity of formulation no. 34 is shown in Fig. 15 and luciferase expression of formulation no. 34 is shown in Fig. 16.

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Claims (18)

  1. Komposition von Plasmid-Liposom-Komplexen zur Benutzung beim Transfizieren einer Wirtszelle mit einem in einem Plasmid enthaltenen Gen, umfassend:
    kondensierte Plasmid-Moleküle, diese Moleküle kondensiert mit einer polykationischen kondensierenden Reagenz und suspendiert in einem wässrigen Medium umfassend eine nicht-elektrolytische Substanz, dieses Medium umfasst eine ionische Stärke geringer als die ionische Stärke einer 10mM Lösung eines monovalenten ionisierbaren Salzes, und
    kationische Liposome umfassend eine Diacylkette, kationisch bläschenformendes Lipid, Dimethyldioctadecylammoniumbromid (DDAB), N[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylämmoniumbromid (DMRIE), N[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromid (DORIE), oder N[1-(2,3,-dioleyloxy) propyl]-N,N,N-trimethylammoniumchlorid (DOTMA), worin diese Komplexe ein Verhältnis von Liposom-Lipid zu Plasmid größer als 5 nmol Liposom-Lipid/µg Plasmid haben und weniger als 25 nmol Liposom-Lipid/µg Plasmid und eine homogene Größe von weniger als 200 nm haben.
  2. Eine Komposition nach Anspruch 1, worin die kondensierten Plasmid-Moleküle DNA-Plasmid-Moleküle sind umfassend ein Gen selektiert aus Genen, welche den Regulator für transmembrane Leitfähigkeit bei zystischer Fibrose, Factor VIII, Interleukin-2 oder p53 kodieren.
  3. Eine Komposition nach einem der Ansprüche 1 oder 2, worin die kondensierende Reagenz ein Polykation ist selektiert aus Histonproteinen, Poly-1-Glutaminen, Protaminen, Melittin und Polymyxin B.
  4. Eine Komposition nach Anspruch 3, worin die kondensierende Reagenz ein Histonprotein ist selektiert aus Totalhiston, Histon 1 und Histon 4.
  5. Eine Komposition nach einem der vorhergehenden Ansprüche, worin das Verhältnis von Liposom-Lipid zu Plasmid zwischen 8 und 18 nmol Liposom-Lipid/µg Plasmid liegt.
  6. Eine Komposition nach einem der vorhergehenden Ansprüche, worin diese gelöste Substanz selektiert wird aus Glukose, Saccharose und Dextran.
  7. Eine Komposition nach einem der vorhergehenden Ansprüche, worin die Diacylkette kationisch bläschenformendes Lipid 1,2-dioleyloxy-3-(trimethylamino)Propan (DOTAP) ist.
  8. Eine Komposition nach einem der vorhergehenden Ansprüche, worin die kationischen Liposome weiter ein neutrales bläschenformendes Lipid umfassen.
  9. Eine Komposition nach Anspruch 8, worin die kationischen Liposome des Weiteren Cholesterin umfassen.
  10. Eine Komposition nach Anspruch 9, worin die kationischen Liposome von DDAB und Cholesterin erstellt werden.
  11. Eine Komposition nach einem der vorhergehenden Ansprüche, worin die kationischen Liposome eine Oberflächenummatelung aus hydrophilen Polymerketten haben, indem ein bläschenformendes Lipid-Derivat mit solch einem hydrophilen Polymer umfasst wird.
  12. Eine Komposition nach Anspruch 11, worin mindestens ein Teil des hydrophilen Polymers an dem bläschenbildenden Lipid angeschlossen ist durch eine Bindung effektif, um die hydrophile Polymerketten freizusetzen, als Antwort auf eine bestehende oder eine induzierte physiologische Bedingung.
  13. Eine Komposition nach entweder Anspruch 11 oder 12, worin die Plasmid-Liposom-Komplexe des Weiteren einen Ligand umfassen, welcher am Distalende der hydrophilen Polymerkette angebracht ist, für Ligand-spezifische Bindungen zu einem Rezeptormolekül auf einer Zell-Zieloberfläche.
  14. Eine Komposition nach einem der Ansprüche 11 bis 13, worin das hydrophile Polymer Polyethylenglycol ist.
  15. Eine Komposition aus Plamid-Liposom-Komplexen nach einem der vorhergehenden Ansprüche zur Verwendung beim Tranfizieren einer Wirtszelle in die Lunge eines Subjekts mit einem DNS-Plasmid umfassend ein Gen selektiert aus Genen, welche den Regulator für transmembrane Leitfähigkeit bei zystischer Fibrose, Interleukin-2 und p53 kodieren.
  16. Eine Komposition aus Plasmid-Liposom-Komplexen nach einem der vorherigen Ansprüche zur Benutzung zum Tranfizieren einer Wirtszelle in einen Tumor in einem Subjekt mit einem DNS-Plasmid umfassend ein Gen selektiert aus Genen, welche für Interleukin-2 und p53 kodieren.
  17. Eine Methode zur Vorbereitung von Plasmid-Liposom-Komplexen umfassend die Schritte:
    - Selektieren als kondensierende Reagenz zum Kondenzieren der Plasmid-Moleküle, ein polykation selektiert aus Histonen, Poly-1-Glutamin, Protamin, Melittin oder Polymxin B;
    - Selektieren eines Mediums, um diese kondensierten Plasmid-Moleküle zu suspendieren, ein wässriges Medium umfassend eine nicht-elektrolytische gelöste Substanz, dieses Medium umfasst eine ionische Stärke geringer als die ionische Stärke von einem 10mM Solutior eines monovalenten ionisierbaren Salzes;
    - Selektieren eines Verhältnisses von Liposom-Lipid zu Plasmid größer als 5nmol Liposom-Lipid/µg Plasmid und weniger als 25 nmole Liposom-Lipid/µg Plasmid; worin die Plasmid-Liposom-Komplexe eine Homogene Größe von weniger als 200nm haben;
    - Kondensieren dies Plasmid-Moleküls; und Mischen des kondensierten Plasmids mit einer Suspension aus kationischen Liposomen umfassend eine Diacylkette kationisch bläschenbildendes Lipid, Dimethyldioctadecylammoniumbromid (DDAB), N[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromid (DMRIE), N[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromid (DORIE), or N[1-(2,3,-dioleyloxy) propyl]-N,N,N-trimethylammoniumchlorid (DOTMA), um diese Plamid-Liposom-Komplexe zu formen.
  18. Eine Methode nach Anspruch 17, um einen Plamid-Liposom Komplex vorzubereiten nach einem der Ansprüche 1 bis 16.
EP98949342A 1997-03-28 1998-09-11 Kondensierte plasmid-liposome komplex zur transfektion Expired - Lifetime EP1109926B1 (de)

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CA2342292A1 (en) 2000-03-23
EP1109926A1 (de) 2001-06-27
ATE362991T1 (de) 2007-06-15
DE69837819T2 (de) 2008-01-31
DE69837819D1 (de) 2007-07-05
US5851818A (en) 1998-12-22
WO2000015825A1 (en) 2000-03-23
AU9568698A (en) 2000-04-03
ES2286857T3 (es) 2007-12-01
HK1039633A1 (en) 2002-05-03

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